在大腸直腸癌腫瘤組織中人類乳突病毒16型E6致癌蛋白表現與p53抑癌基因失活之關聯性

Human papilloma virus 16 E6 oncoprotein associated with p53 inactivation in colorectal cancer

陳丹霞(Tan-Hsia Chen)

指導教授 ： 林俊哲

中山醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00113

摘要

高危險性的人類乳突病毒(human papillomavirus, HPV) 16/18之感染被認為可能參與子宮頸癌及肛門癌的形成，而和非生殖器癌症的口咽癌也有相關聯。不過，其他非生殖器癌症(例如：肺癌、乳癌、大腸直腸癌)和HPV的相關性並不清楚。雖然HPV感染後可經血液循環跑到身體的其他器官，但仍然缺乏足夠的証據闡明HPV如何傳染到內臟。
大腸直腸癌位居世界癌症死亡排名之第三位，它的危險因子相關研究主要是抽煙、發炎性大腸炎症、紅肉攝取等，亦有些研究推測高危險性之人類乳突病毒(human papillomavirus, HPV) 16/18可能參與大腸直腸癌的形成。在過去的二十年，大量的醫學研究發現HPV和大腸直腸癌有相關。這些研究指出HPV16型是主要發生在大腸直腸癌的亞型，然而HPV和大腸直腸癌的關係仍存在不一致的結論。主要的原因是很多研究只要在大腸癌檢體上偵測到HPV DNA就認為HPV和大腸直腸癌有關係，卻缺乏証據指出HPV參予了大腸直腸癌的致癌機轉。
因此本研究收集 69位大腸直腸癌之腫瘤組織，分別以 nested-PCR和in situ hybridization (ISH)分析HPV16是否感染大腸直腸腫瘤組織以及是否感染腫瘤組織周邊中之正常細胞，尤其是血管內皮細胞和淋巴細胞等，以了解HPV感染至大腸直腸腫瘤組織是否經由血液循環。結果發現以nested-PCR方法分析69位大腸直腸腫瘤患者中，有11位患者之大腸直腸腫瘤組織具HPV 16 DNA，其陽性率為16% (11/69)。本研究進一步以ISH方法確定HPV 16 DNA之感染，結果發現有8/11(73%)位患者之腫瘤組織具有HPV 16 DNA的存在。在我們的研究中，HPV 16 DNA是否存在於大腸直腸腫瘤組織和病患的臨床資料(年齡、性別、抽煙、腫瘤位置)皆無關係。但在第一期大腸直腸癌組織中感染HPV16的比例多於其他期別(II, III, IV)。我們接著探討HPV16是否參予大腸直腸癌的癌化過程。本研究以免疫組織化學染色法(immunohistochemistry, IHC)分析這些11位ISH確認存在HPV16 DNA大腸直腸癌的腫瘤組織，結果發現有8位患者之腫瘤組織的病理連續切片呈HPV 16 E6致癌蛋白表現。再者，我們觀察到在E6致癌蛋白陽性的腫瘤其鄰近的正常組織細胞(例如血管內皮細胞、淋巴球、纖維母細胞和腺細胞等)也有E6致癌蛋白表現。根據過去的研究，已知HPV引起子宮頸癌的主要致癌路徑是表現E6致癌蛋白將抑癌基因去活化。因此本研究為了進一步了解HPV 16感染是否參與大腸直腸癌之癌化過程，本研究以IHC分析偵測這8位患者之腫瘤組織的病理切片中之HPV 16 E6致癌蛋白和p53表現之相關性。結果發現在4位E6致癌蛋白陽性且p53 wild-type大腸直腸腫瘤組織中有3(3/4，75%)位患者之p53蛋白不表現，而1位是弱p53免疫染色反應。另外其他4位患者之腫瘤組織有E6致癌蛋白表現，同時亦能偵測到p53蛋白表現，經過p53 DNA序列分析之結果顯示，這些患者之p53發生基因突變，因而p53蛋白沒有被E6致癌蛋白降解。
我們進一步探討是否E6致癌蛋白(不管是否具有p53基因突變)會減少p53轉錄活性。為了分析是否E6致癌蛋白陽性和p53突變會減少p53轉錄活性，根據以往對p53抑癌基因之瞭解，進一步分析p53的下游基因mdm2 mRNA和p21蛋白表現，結果使用Real-time RT-PCR分析指出E6致癌蛋白陽性/p53-wild type的腫瘤，其p21及mdm2 mRNA的蛋白表現顯著低於鄰近的正常組織;而在E6致癌蛋白陽性/p53突變的腫瘤，其p21及mdm2 mRNA的表現也低於鄰近的正常組織。這些結果指出E6致癌蛋白在那些沒有p53突變的腫瘤是經由p53去活化而減少了p21及mdm2蛋白的表現。由以上之結果推測HPV 16感染至大腸直腸組織，可經由血液循環而造成，同時發現HPV 16參與一些大腸直腸癌患者之癌化過程，可能經由表現E6致癌蛋白抑制p53路徑所致。

關鍵字

人類乳突病毒 ； 大腸直腸癌 ； E6致癌蛋白 ； p53抑癌基因

並列摘要

Background: The aim of this study is to investigate the association between human papilloma virus (HPV) infection and colorectal cancer. Colorectal cancer is the third place of cancer death in the world. Most etiological studies of this disease are focused on dietary factors and cigarette smoking behavior. A few reports indicate the association between high risk HPV and colorectal cancer. The association of Human papillomavirus (HPV) infection with colorectal cancer has been extensively investigated, but no available evidence supports the involvement of HPV in colorectal cancer development.

Materials and Methods: Sixty-nine patients with pathologically confirmed primary colorectal cancer including 6 stage I, 24 stage II, 21 stage III, and 18 stage IV patients were enrolled in this study to investigate whether HPV 16 could be involved in colorectal tumorigenesis. Nested-polymerase chain reaction (nested-PCR) was used to detect HPV16 DNA in colorectal tumor tissues and further confirmed by in situ hybridization (ISH). In addition, immunohistochemistry analysis was performed to examine the E6 oncoprotein in colorectal tumors. To verify whether E6 could inactivate the p53 transcriptional function, the levels of p21 and Mdm2 mRNA expression were evaluated by real-time reverse transcription (RT)-PCR.

Results: Of the 69 colorectal tumors, HPV16 DNA was detected in 11 (16%) by nested-PCR, and HPV16 DNA was present in 8 of the 11 (73%) tumors which was confirmed by ISH. The presence of HPV16 DNA in colorectal tumors was not associated with patients’ clinical parameters including age, gender, smoking status, tumor site; however, HPV16 infection was more common in stage I patients than in late-stage patients (II, III, and IV). We next asked whether HPV16 infection could be linked with colorectal cancer development. Immunohistochemical data indicated that 8 of the 11 HPV16 DNA-positive tumors had E6 oncoprotein expression. Moreover, we also observed that the adjacent normal tissues including endothelial cells, lymphocytes, fibroblasts, and gland cells in E6-positive tumors had E6oncoprotein expression. In addition, 3 of the 4 (75%) E6-positive tumors carrying p53 wild-type had negative immunostaining, but one tumor had less p53 immunostaining. We further examined whether E6-positive and/or p53 mutated tumors reduce p53 transcriptional activity. Real-time RT-PCR analysis indicated that p21 and mdm2 mRNA expression levels in E6/p53-wildtype tumors were significantly lower than in their adjacent normal tissues; as expected, E6-positive/p53-mutated tumors had lower p21 and mdm2 mRNA expression levels compared with their adjacent normal tissues. These results clearly indicate that the E6 oncoprotein expressed in p53 wildtype tumors may reduce p21 and mdm2 expression via p53 inactivation.

Conclusion: These results suggest that HPV16 infection may be involved in a subset of colorectal cancer, and we suggest that the transmission of HPV to the colon and rectum might occur through peripheral blood lymphocytes.

並列關鍵字

Human papilloma virus ； colorectal cancer ； E6 oncoprotein ； p53

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